Cyclic gnrh antagonists

ABSTRACT

Peptides which inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. These peptides may be used to treat steroid-dependent tumors, such as prostatic and mammary tumors. The peptides include cyclic, bicyclic and tricyclic analogs of the decapeptide GnRH, and preferably there are at least two covalent bonds between the residues in the 4- and 10-positions, the residues in the 5- and 8-positions and the residues in the 1- and 3-positions, respectively. Examples of such bonds include a disulfide linkage between Cys residues, an amide linkage between a side chain amino group and a side chain carboyxl group, a dicarba linkage between side-chain alkyl groups, and a carba linkage between a side-chain alkyl group and a side-chain sulfhydryl group.

This invention was made with Government support under Grant HD-02903-B awarded by the National Institutes of Health (DHHS). The Government has certain rights in this invention.

The present invention relates to peptides which inhibit the release of gonadotropins by the pituitary gland in mammalians, including humans, and to methods of preventing ovulation and/or inhibiting the release of steroids More particularly, the present invention is directed to peptides which inhibit gonadal function and the release of the steroidal hormones, progesterone and testosterone.

BACKGROUND OF THE INVENTION

The pituitary gland is attached by a stalk to the region in the base of the brain known as the hypothalamus. In particular, follicle stimulating hormone (FSH) and luteinizing hormone (LH), sometimes referred to as gonadotropins or gonadotropic hormones, are released by the pituitary gland. These hormones, in combination, regulate the functioning of the gonads to produce testosterone in the testes and progesterone and estrogen in the ovaries, and they also regulate the production and maturation of gametes.

The release of a hormone by the anterior lobe of the pituitary gland usually requires a prior release of another class of hormones produced by the hypothalamus. One of the hypothalamic hormones acts as a factor that triggers the release of the gonadotropic hormones, particularly LH, and this hormone is referred to herein as GnRH although it has also been referred to as LH-RH and as LRF. GnRH has been isolated and characterized as a decapeptide having the following structure:

    pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2

Peptides are compounds which contain two or more amino acids in which the carboxyl group of one acid is linked to the amino group of the other acid. The formula for GnRH, as represented above, is in accordance with conventional representation of peptides where the amino terminus appears to the left and the carboxyl terminus to the right The position of the amino acid residue is identified by numbering the amino acid residues from left to right. In the case of GnRH, the hydroxyl portion of the carboxyl group of glycine at the C-terminus has been replaced with an amino group(NH₂). The abbreviations for the common individual amino acid residues are conventional and are based on the trivial name of the amino acid, e.g. Pglu is pyroglutamic acid, Glu is glutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Nle is norleucine, Orn is ornithine, Arg is arginine, Pro is proline, Sar is sarcosine, Phe is phenylalanine, Ala is alanine, Val is valine, Nva is norvaline, Ile is isoleucine, Thr is threonine, Lys is lysine, Asp is aspartic acid, Asn is asparagine, Gln is glutamine, Cys is cysteine, and Met is methionine. Except for glycine, the amino acids which appear in the peptides of the invention are of the L-configuration unless noted otherwise.

There are reasons for desiring to prevent ovulation in female mammalians, and the administration of GnRH analogs that are antagonistic to the normal function of GnRH have been used to suppress or delay ovulation. For this reason, analogs of GnRH which are antagonistic to GnRH are being investigated for their potential use as a contraceptive or for regulating conception periods. GnRH antagonists may also be used for the treatment of precocious puberty and endometriosis. Such antagonists have also been found useful to regulate the secretion of gonadotropins in male mammals and can be employed to arrest spermatogenesis, e.g. as male contraceptives, and for treatment of prostatic hypertrophy. More specifically, GnRH antagonists can be used to treat steroid-dependent tumors, such as prostatic, brain and mammary tumors.

It is desired to provide improved peptides which are strongly antagonistic to endogenous GnRH and which prevent secretion of LH and the release of steroids by the gonads of mammals, and to also provide compounds which are biologically effective in vivo when administered orally.

SUMMARY OF THE INVENTION

The present invention provides peptides which inhibit the release of gonadotropins in mammalians, including humans, and it also provides methods for inhibiting the release of steroids by the gonads of male and female mammalians. The improved GnRH analogs are strongly antagonistic to GnRH and have an inhibitory effect on the reproduction processes of mammalians; thus, they are referred to as GnRH antagonists. These analogs may be used to inhibit the production of gonadotropins and sex hormones under various circumstances, including precocious puberty, hormone dependent neoplasia, dysmenorrhea, endometriosis and steroid-dependent tumors.

Generally, in accordance with the present invention, peptides have been synthesized which strongly inhibit the secretion of gonadotropins by the pituitary gland of mammalians, including humans, and/or inhibit the release of steroids by the gonads. These peptides are cyclic, and are preferably bicyclic or tricyclic analogs of GnRH wherein there are at least two covalent bonds, i.e., between the following pairs of residues: the 4-position residue and the 10-position residue, the 5-position residue and the 8-position residue and the 1-position residue and the 3-position residue. Certain bicyclic peptides which do not have a covalent bond between the 1- and 3-position residues should have a 1-position substitution, preferably dehydroPro or β-(1-or 2-naphthyl)-D-alanine(hereinafter β-D-1NAL or β-D-2NAL) and a 3-position substitution, preferably in the form of unsubstituted or substituted D-Trp, D-3PAL, β-D-2NAL or β-D-1NAL. All of the peptides preferably have a 2-position substitution in the form of a modified D-Phe residue. The 4-position substitution may be Cys, a diamino acid having not more than 5 carbon atoms, a dicarboxyl amino acid, such as Asp or Glu, or Abu. The 5-position is preferably occupied by Glu and the 8-position by Lys when these two residues are covalently bonded; however, optionally the following residues can be employed in these positions: homoglutamic acid (Hgl); homohomoglutamic acid (Hhg), i.e., 1,7-dicarboxy, 2-amino, heptanoic acid; homolysine (Hly) and homohomolysine (Hhl) or 1-carboxy, 2,8-diamino, octanoic acid. The peptide also has a 6-position substitution, can have an optional substitution in the 7-position, such as Nle, NML, Phe, Nva, Met, Tyr, Trp or PAL, and can have an optional modification of Pro in the 9-position. The substitution in the 10-position is complementary to the 4-position residue. For the tricyclic compounds and certain of the bicyclic compounds, the residues in the 1- and 3-positions are complementary, as generally set forth with respect to those in the 4- and 10-positions; however, it may be preferred to employ a dicarba bond between these two residues of a length longer than that provided by a pair of Abu residues.

Modified D-Phe in the 2-position provides increased antagonistic activity as a result of the specific modifications present in the benzene ring. Single substitutions for hydrogen in the ring are preferably made in the para- or 4-position, but might instead be in either the 2- or 3-position; the substitutions are selected from chloro, fluoro, bromo, methyl, methoxy and nitro, with chloro, fluoro and nitro being preferred. Dichloro substitutions are in the 2,4 or 3,4 positions in the ring. The alpha-carbon atom may also be methylated, e.g. (C^(a) Me/4Cl)Phe. The 1-position substituent is preferably modified so that its alpha amino group contains an acyl group, such as formyl(For), acetyl(Ac), acrylyl(Acr), vinylacetyl(Vac) or benzoyl(Bz), with acetyl and acrylyl being preferred. PAL and D-PAL represent the L- and D-isomers of pyridylalanine where the β-carbon of Ala is linked to the 2-, 3- or 4-position, preferably to the 3-position, on the pyridine ring. When β-D-NAL is present in the 1-position, a hydrophillic D-amino acid residue, such as 4NH₂ -D-Phe, 4-guanidino-D-Phe, D-His, D-Arg, D-Har(Homoarginine) or D-PAL is preferably present in the 6-position. When dehydroPro is present in the 1-position, a D-isomer of a lipophilic amino acid, such as D-Trp, D-Phe, For-D-Trp, NO₂ -D-Trp, D-Leu, D-Ile, D-Nle, D-Tyr, D-Val, D-Ala, D-Ser(OtBu), β-D-NAL or (imBzl)D-His is preferably in the 6-position, but D-PAL may be used.

These GnRH antagonists are active when administered orally compared to previously available GnRH antagonists. The peptides inhibit ovulation of female mammals when administered at low levels at proestrus and are also effective to cause resorption of fertilized eggs if administered shortly after conception. These peptides are also effective for the contraceptive treatment of male mammals and for the treatment of steroid-dependent and other tumors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

More specifically, certain preferred bicyclic peptides of the present invention are represented by the following Formula I: ##STR1## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁ is dehydroPro, D-pGlu, (A)D-Phe, (B)D-Trp, Pro, or β-D-NAL; A is H, Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; R₃ is D-PAL, β-D-NAL or (B)D-Trp; R₄ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu) or D-Nva; A' is A, NH₂, NHCH₃ or gua; C is H, imBzl or dinitrophenol; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; E is H, OH or dehydro and R₁₀ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R₄ is Cys or Abu, R₁₀ is Cys or Abu; when R.sub. 4 is Asp or Glu, R₁₀ is Orn, Dbu or Dpr; and when R₄ is Orn, Dbu or Dpr, R₁₀ is Asp or Glu. When R₁ is β-D-NAL, then R₆ is preferably 4-NH₂ -D-Phe, D-Har, D-His, 4-gua-D-Phe, D-PAL or D-Arg. As indicated hereinbefore, Hgl or Hhg can be substituted for Glu in the 5-position, and Hly or Hhl can be substituted for Lys in the 8-position.

By dehydroPro is meant 3,4 dehydroproline, C₅ H₇ O₂ N. By β-D-NAL is meant the D-isomer of alanine which is substituted by naphthyl on the β-carbon atom, i.e., also 3-D-NAL. Preferably β-D-2NAL is employed, the attachment to naphthalene is at the 2-position on the ring structure; however, β-D-1NAL may also be used. PAL represents alanine which is substituted by pyridyl on the β-carbon atom; preferably the linkage is to the 3-position on the pyridine ring. When substituted D-Trp is employed, single substitutions for hydrogen may be made in either the 5- or 6-position, which are selected from chloro, fluoro, bromo, methyl, amino, methoxy and nitro, with chloro, fluoro and nitro being preferred. Alternatively, the indole nitrogen may be acylated, e.g. with formyl (N^(in) For- or 1For-) or with acetyl. N^(in) For-D-Trp and 6NO₂ -D-Trp are the preferred substituted residues. When either D-Arg or D-Har is present in the 6-position, or Arg is present in the 8-position, the guanidino side chain can be di-substituted with lower alkyl(C₁ to C₄), preferably diethyl. By NML is meant N^(a) CH₃ -L-Leu. By Abu is meant 2-aminobutyric acid and by Dbu is meant 2,4-diaminobutyric acid. By Dpr is meant 2,3-diaminopropionic acid. When dehydroPro is present in the 1-position, a lipophilic residue is preferably in the 6-position. By 4-gua-D-Phe is meant a residue of D-Phe having a guanidino group substituted in the para-position.

Other biologically active monocyclic and bicyclic peptides can be fashioned which have a covalent bond between the residues in the 1- and 3-positions and may optionally have such a bond between the residues in the 4- and 10-positions or between the residues in the 5- and 8-positions. Such peptides may have the following formula, with the understanding that there would be a cyclizing covalent bond between residues R₁ and R₃, and optionally one between R₅ and R₈, or between R₄ and R₁₀ : ##STR2## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁ is D-Cys, D-Abu, asp, glu, hgl, hhg, lys, hly, hhl, orn, dbu or dpr; A is H,Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; R₃ is D-Cys, abu, asp, glu, hgl, hhg, lys, hly, hhl, orn, dbu or dpr; R₄ is Ser, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₅ is Tyr or Glu; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; C is H, imBzl or dinitrophenol; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; E is H, OH or dehydro; R₈ is (D)Arg, (D)Har or Lys and R₁₀ is Gly, D-Ala, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R₄ is Cys or Abu, R₁₀ is Cys or Abu; when R₄ is Asp or Glu, R₁₀ is Orn, Dbu or Dpr; when R₄ is Orn, Dbu or Dpr, R₁₀ is Asp or Glu; when R₅ is Glu, R₈ is Lys; and provided further that when R₁ is D-Cys or D-Abu, R₃ is D-Cys or D-Abu; when R₁ is asp, glu, hgl or hhg, R₃ is lys, hly, hhl, orn, dbu or dpr; and when R₁ is lys, hly, hhl, orn, dbu or dpr, R₃ is asp, glu, hgl or hhg. By the lower case designation, e.g. asp, is meant that the residue may be either the L- or the D-isomer, i.e. L-Asp or D-Asp; however, the D-isomers are preferred in both the 1- and 3-positions.

In certain preferred tricyclic peptides, the formula is generally as set forth above except that R₁ is D-Cys, D-Abu, D-Asp, D-Glu, D-Hgl, D-Hhg, D-Lys, D-Hly, D-Hhl, D-Orn, D-Dbu or D-Dpr and R₃ is a complementary residue selected from the same group; however, in the tricyclic peptides at least 2 of the 3 cyclizing bonds should preferably be amide and/or dicarba bonds. The following formula is representative of certain particularly preferred tricyclic peptides: ##STR3## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁ is D-Cys, D-Abu, D-Asp, D-Glu, D-Hgl, D-Hhg, D-Lys, D-Hly, D-Hhl, D-Orn, D-Dbu or D-Dpr; A is H, Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; R₃ is D-Cys, D-Abu, D-Asp, D-Glu, D-Hgl, D-Hhg, D-Lys, D-Hly, D-Hhl, D-Orn, D-Dbu or D-Dpr; R₄ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; B is H,NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; C is H, imBzl or dinitrophenol; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; E is H, OH or dehydro and R₁₀ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R₄ is Cys or Abu, R₁₀ is cys or Abu; when R₄ is Asp or Glu, R₁₀ is Orn, Dbu or Dpr; and when R₄ is Orn, Dbu or Dpr, R₁₀ is Asp or Glu and provided further that when R₁ is D-Cys or D-Abu, R₃ is D-Cys or D-Abu; when R₁ is D-Asp, D-Glu, D-Hgl or D-Hhg, R₃ is D-Lys, D-Hly, D-Hhl, D-Orn, D-Dbu or D-Dpr; and when R₁ is D-Lys, D-Hly, D-Hhl, D-Orn, D-Dbu or D-Dpr, R₃ is D-Asp, D-Glu, D-Hgl or D-Hhg.

It may be desirable to have a longer cyclizing linkage between the two residues in the 1- and 3-positions than the dicarba linkage between two D-Abu residues, and in such an instance, peptides may be synthesized having the formula: ##STR4## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; A is H,Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; n=4 to 11; R₄ is Ser, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₅ is Tyr, Glu, Hgl or Hhg; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; C is H, imBzl or dinitrophenol; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; E is H, OH or dehydro; R₈ is (D)Arg, (D)Har, Lys, Hly or Hhl and R₁₀ is Gly, D-Ala, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R₄ is Cys or Abu, it is bonded to R₁₀ which is Cys or Abu; when R₄ is Asp or Glu, it is bonded to R₁₀ which is Orn, Dbu or Dpr; when R₄ is Orn, Dbu or Dpr, it is bonded to R₁₀ which is Asp or Glu; and when R₅ is Glu, Hgl or Hhg, it is bonded to R₈ which is Lys, Hly or Hhl. When the residues in positions 1 and 3 are both Abu, n=4; however, preferably, n=7 to 9. Such peptide may be monocyclic, dicyclic or tricyclic.

The peptides of the present invention can be synthesized by classical solution synthesis or by a solid phase technique using a chloromethylated resin, a hydroxymethylated resin, a methylbenzhydrylamine resin (MBHA), a benzhydrylamine (BHA) resin or any other suitable resin known in the art. The solid phase synthesis is conducted in a manner to stepwise add the amino acids in the chain in the manner set forth in detail in the U.S. Pat. No. 4,211,693. Side-chain protecting groups, as are well known in the art, are preferably added to the residues to be employed in the synthesis having particularly labile side chains and may optionally be added to others, such as Trp, before these amino acids are coupled to the chain being built upon the resin. Such synthesis provides the fully protected intermediate peptidoresin.

Chemical intermediates made generally in accordance with the invention to produce the preferred bicyclic peptides may be represented by Formula II:

    X.sup.1 -R.sub.1 -(A)D-Phe-R.sub.3 (X.sup.2)-R.sub.4 (X.sup.3)-R.sub.5 (X.sup.6)-R.sub.6 (X.sup.4 or X.sup.5)-R.sub.7 (X.sup.2 or X.sup.4 or X.sup.7)-R.sub.8 (X.sup.8)-(E)Pro-R.sub.10 (X.sup.3)-X.sup.9

wherein: X¹ is an alpha-amino protecting group of the type known to be useful in the art in the stepwise synthesis of polypeptides and when X in the desired peptide composition is a particular acyl group, that group may be used as the protecting group. Among the classes of alpha-amino protecting groups covered by X¹ are (1) acyl-type protecting groups, such as formyl(For), trifluoroacetyl, phthalyl, p-toluenesulfonyl(Tos), benzoyl(Bz), benzenesulfonyl, o-nitrophenylsulfenyl(Nps), tritylsulfenyl, o-nitrophenoxyacetyl, acrylyl(Acr), chloroacetyl, acetyl(Ac) and γ-chlorobutyryl; (2) aromatic urethan-type protecting groups, e.g., benzyloxycarbonyl(Z), fluorenylmethyloxycarbonyl(Fmoc) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxy-carbonyl(ClZ), p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as tertbutyloxycarbonyl(Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6) alkyl-type protecting groups, such as allyl(Aly), triphenylmethyl(trityl) and benzyl(Bzl); (7) trialkylsilane groups, such as trimethylsilane. The preferred alpha-amino protecting group is Boc, particularly when X is hydrogen.

X² is hydrogen or a suitable protecting group for the indole nitrogen of Trp, such as Bz.

X³ is a protecting group for the sulfhydryl group of Cys, preferably p-methoxybenzyl(MeOBzl), p-methylbenzyl, acetamidomethyl, trityl or Bzl; or a suitable protecting group for an amino side chain, e.g. 2Cl-Z or t-amyloxycarbonyl; or a suitable, preferably hydrazine-labile, protecting group for a carboxyl side chain, such as OBzl(benzyl ester); or is a direct bond where the cyclic form results from a carba or dicarba bond.

X⁴ is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzyl, Z, 2-bromobenzyloxycarbonyl (2BrZ) and 2,6-dichlorobenzyl(DCB). 2BrZ is preferred.

X⁵ is a protecting group for a side chain guanidino group, such as that in Arg, or for the imidazole group of His, such as nitro, Tos, trityl, adamantyloxycarbonyl, Z and 2,4-dinitrophenyl(DNP), or X⁵ may be hydrogen, which means there is no protection on the side chain group atoms. Tos is generally preferred.

X⁶ is hydrogen or a base-labile protecting group for a side-chain carboxyl group, preferably fluorenylmethyl ester (OFm), or is a covalent bond.

X⁷ is hydrogen or a protecting group for Met, such as oxygen.

X⁸ is a base-labile protecting group for an amino side chain group, preferably Fmoc, or is a covalent bond.

X⁹ may be O-CH₂ -[resin support], -NH-[resin support], OH, ester or NH₂.

The criterion for selecting side-chain protecting groups for X² -X⁸ is that the protecting group should be stable to the reagent under the reaction conditions selected for removing the alpha-amino protecting group (preferably Boc) at each step of the synthesis. The protecting group should not be split off under coupling conditions but should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain. The protecting groups X⁶ and X⁸ in some syntheses should be removable without the removal of the protecting group X³.

When the X⁹ group is -O-CH₂ -[resin support], the ester moiety of one of the many functional groups of a polystyrene resin support is being represented. When the X⁹ group is -NH-[resin support], an amide bond connects R₁₀ to a BHA resin or to a MBHA resin.

When X is acetyl, for example, in the final formula, it may be possible to employ it as the X¹ protecting group for the alpha-amino group of D-NAL or whatever amino acid is used in the 1-position by adding it before the coupling of this last amino acid to the peptide chain. However, a reaction is preferably carried out with the peptide on the resin (after deblocking the alpha-amino group while the side-chain groups remain protected), e.g. by reacting with acetic acid in the presence of dicyclohexyl carbodiimide (DCC) or preferably with acetic anhydride or by another suitable reaction as known in the art.

In making the preferred bicyclic peptides, one cyclization is preferably effected of the 6-residue peptide intermediate while it is a part of the peptidoresin. After this cyclization is effected, the synthesis of the decapeptide is completed. If a tricyclic peptide is being synthesized or if an amido bond is being established between the 1- and 3-position residues, cyclization may be carried out on the resin between the 1- and 3-position residues. The protected peptide is then suitably cleaved from the resin, e.g., from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, or alternatively, from a benzhydrylamine resin with hydrofluoric acid (HF) causing deprotection as well as cleavage of the peptide.

The final cyclizing steps for the GnRH peptide analog depend, of course, upon the type of linkage which is intended between the residues in the 4- and 10-positions; they should also take into consideration the amide bond linkage between the residues in positions 5 and 8 as well as any linkage which optionally may have been formed between the 1- and the 3-position residues. It is generally preferred to carry out the final cyclizing step to form the bond between the position 4- and 10-residues following the cleavage from the resin. Such a disulfide form of bond is obtained by oxidizing using a ferricyanide solution, preferably as described in Rivier et al., Biopolymers, Vol. 17 (1978), 1927-38, or by air oxidation, or in accordance with other known procedures.

When the cyclization is via an amide bond between a side-chain amino group of the 4-position residue and a side-chain carboxyl group of the 10-position residue (which may be preferred), or vice-versa, it is preferable to synthesize the protected peptide on an MBHA or BHA resin and to derivatize the benzyl ester of the carboxyl acid side chain to the hydrazide while the peptide is still attached to the resin. This can be accomplished by using OBzl as a protecting group for the carboxyl side-chain of the residue to be involved in the amide-bond bridge. Following this selective hydrazide activation, deprotection of the remainder of the side-chain protecting groups and cleavage from the resin are effected. Then reaction to accomplish cyclization is carried out by treating with isoamylnitrite and a strong acid, such as HCl, to generate the azide which in turn reacts with the free amino group on the side chain of the 10-position residue, after a neutralization step, generating the amide bond.

Analogs of GnRH including the equivalent of modified cysteine residues in the 4- and 10-positions wherein the disulfide linkage has been replaced by --CH₂ -- linkage are referred to as dicarba. If only one of the sulfhydryl groups is replaced by a CH₂ -group, it is referred to as carba, e.g., [carba⁴, Cys¹⁰ ]-GnRH. In general, cyclizations of peptides in this overall fashion are exemplified by the teachings of the following U.S. Pat. Nos. 4,115,554, (Sept. 19, 1978); 4,133,805 (Jan. 9, 1979); 4,140,767 (Feb. 20, 1979); 4,161,521 (July 17, 1979); 4,191,754 (Mar. 4, 1980); 4,238,481 (Dec. 9, 1980); 4,244,947 (Jan. 13, 1981); and 4,261,885 (Apr. 14, 1981). Viewed from the aspect of the ultimate peptide, the location which would otherwise have been occupied by a Cys residue instead contains a residue of alpha-amino butyric acid(Abu). When preparing peptides having such a dicarba or carba-S linkage or a longer chain dicarba linkage, the procedure set forth in either U.S. Pat. No. 4,161,521 or U.S. Pat. No. 4,703,106 may be employed (the disclosures of which patents are incorporated herein by reference); in either case, in the intermediate of Formula II, X³ is a direct bond to the other residue.

Thus, for example, the invention also provides a method for making certain preferred bicyclic peptide or a nontoxic salt thereof, which peptides have the Formula I: ##STR5## wherein X is hydrogen or an acyl group having ; or less carbon atoms; R₁ is dehydroPro, D-pGlu, (A)D-Phe, (B)D-Trp, Pro, or β-D-NAL; A is H,Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; R₃ is D-PAL, β-D-NAL or (B)D-Trp; R₄ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; C is H, imBzl or di nitrophenyl; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; E is H, OH or dehydro and R₁₀ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R₄ is Cys or Abu, R₁₀ is Cys or Abu; when R₄ is Asp or Glu, R₁₀ is Orn, Dbu or Dpr; and when R₄ is Orn, Dbu or Dpr, Re is Asp or Glu; which method comprises (a) forming an intermediate compound having the Formula II:

    X.sup.1 -R.sub.1 -(A)D-Phe-R.sub.3 (X.sup.2)-R.sub.4 (X.sup.3)-Glu(X.sup.6)-R.sub.6 (X.sup.4 or X.sup.5)-R.sub.7 (X.sup.2 or X.sup.4 or X.sup.7)-Lys(X.sup.8)-(E)Pro-R.sub.10 (X.sup.3)-X.sup.9

wherein X¹ is hydrogen or an alpha-amino protecting group; X² is hydrogen or a protecting group for an indole nitrogen; X³ is a direct bond, hydrogen or a protecting group for Cys or for a side-chain amino or carboxyl group; X⁴ is hydrogen or a protecting group for a phenolic hydroxyl group of Tyr; X⁵ is either hydrogen or a protecting group for a guanidino or imidazole side chain; X⁶ is a base-labile protecting group for a carboxyl side chain or a direct bond; X⁷ is a protecting group for Met; X⁸ is a base-labile protecting group for a side-chain amino or a direct bond; and X⁹ is selected from the group consisting of O--CH₂ --(resin support), --NH--(resin support), esters, and amides; (b) splitting off one or more of the groups X¹ to X⁸ and/or cleaving from any resin support included in X⁹ ; (c) optionally creating a cyclizing bond between R₄ and R₁₀ if not already present and, if desired, (d) converting a resulting peptide into a nontoxic salt thereof. Preferably the intermediate for certain preferred bicyclic peptides has the Formula (III): ##STR6## wherein X⁶ and X⁸ in Formula (II) constitute a direct amide bond. Preferably the intermediate for certain preferred tricyclic peptide has the Formula (IIIA); ##STR7##

Purification of the peptide may be effected by ion exchange chromotography on a CMC column, followed by partition chromotography using the elution system: n-butanol;0.1N acetic acid (1:1 volume ratio) on a column packed with Sephadex G-25; however, it is preferably carried out by using HPLC, as known in the art and set forth specifically in J. Rivier, et al. J. Chromatography, 288 (1984) 303-328.

The peptides of the invention are effective at levels of less than 100 micrograms per kilogram of body weight, when administered at about noon on the day of proestrous, to prevent ovulation in female rats. For prolonged suppression of ovulation, it may be necessary to use dosage levels in the range of from about 0.1 to about 2.5 milligrams per kilogram of body weight. These antagonists are also effective to arrest spermatogenesis when administered to male mammals on a regular basis and can thus be used as contraceptives. Since these compounds will reduce testosterone levels (an undesired consequence in the normal, sexually active male), it may be reasonable to administer replacement dosages of testosterone along with the GnRH antagonist. These antagonists can also be used to regulate the production of gonadotropins and sex steroids for other purposes as indicated hereinbefore.

EXAMPLE I

Bicyclic peptides as indicated in TABLE I having the formula: ##STR8## are prepared by the solid-phase procedure referred to above.

                  TABLE I                                                          ______________________________________                                         R.sub.1    R.sub.3   R.sub.4 R.sub.6   R.sub.10                                ______________________________________                                          1  β-D--2NAL                                                                            D--Trp    Asp   D--Arg    Dpr                                    2  "          "         Dpr   "         Asp                                    3  "          D--3PAL   Asp   β-D--2NAL                                                                           Dpr                                    4  dehydroPro D--Trp    Cys   "         Cys                                    5  β-D--2NAL                                                                            "         "     "         "                                      6  "          D--3PAL   Dpr   D--3PAL   Asp                                    7  "          "         Orn   "         Asp                                    8  dehydroPro β-D2NAL                                                                             Asp   β-D--2NAL                                                                           Dpr                                    9  "          D--3PAL   "     D--Tyr    Dpr                                   10  B-D--2NAL  D--Trp    Glu   D--2PAL   Dpr                                   11  "          "         Dbu   (4gua)D--Phe                                                                             Asp                                   12  "          "         "     "         "                                     13  dehydroPro "         "     D--Ala    Glu                                   14  D--Trp     D--4PAL   "     D--Phe    "                                     15  D--pGlu    D--Trp    Dpr   D--Ile    "                                     16  D--Phe     D--2PAL   "     D--Val    Asp                                   ______________________________________                                    

For purposes of an example, a representative solid phase synthesis of Peptide No. 1 above, which is referred to as (Cyclo 4-10, 5-8) [Ac-β-D-2NAL¹, (4Cl)D-Phe², D-Trp³, Asp⁴, Glu⁵, D-Arg⁶, Lys⁸, Dpr¹⁰ ]-GnRH is set forth hereinafter. This peptide has the following formula: ##STR9## The other peptides are similarly synthesized and purified.

Five grams of a BHA resin are used, and Boc-protected Dpr is coupled to the resin over a 2-hour period in CH₂ Cl₂ using a 3-fold excess of Boc derivative and DCC as an activating reagent. The Dpr residue attaches to the BHA residue by an amide bond.

Following the coupling of each amino acid residue, washing, deblocking and coupling of the next amino acid residue is carried out in accordance with the following schedule, using an automated machine, when beginning with about 5 grams of resin:

    ______________________________________                                                                      MIX TIMES                                         STEP  REAGENTS AND OPERATIONS                                                                               MIN.                                              ______________________________________                                         1     CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                               3                                                 2     Methanol(MeOH) wash-30 ml. (2 times)                                                                  3                                                 3     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                               3                                                 4     50 percent TFA plus 5 percent 1,2-eth-                                                                10                                                      anedithiol in CH.sub.2 Cl.sub.2 -70 ml. (2 times)                        5     Isopropyl alcohol + 1% ethanedithiol                                                                  3                                                       wash-80 ml. (2 times)                                                    6     TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                                                         5                                                       (2 times)                                                                7     MeOH wash-40 ml. (2 times)                                                                            2                                                 8     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                               3                                                 9     Boc-amino acid (10 mmoles) in 30 ml. of                                                                30-300                                                 either DMF or CH.sub.2 Cl.sub.2, depending                                     upon the solubility of the particular                                          protected amino acid, (1 time)                                                 plus DCC (10 mmoles) in CH.sub.2 Cl.sub.2                                10    MeOH wash-40 ml. (2 times)                                                                            3                                                 11    TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                                                         3                                                       (1 time)                                                                 12    MeOH wash-30 ml. (2 times)                                                                            3                                                 13    CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                               3                                                 ______________________________________                                    

Should the synthesis be manually performed, after step 13, an aliquot may be taken for a ninhydrin test. If the test is negative, proceed to step 1 for coupling of the next amino acid; if the test is positive or slightly positive, go back and repeat steps 9 through 13.

The above schedule is used for coupling of each of the amino acids of the peptide of the invention after the first amino acid has been attached. N^(a) Boc protection is used for each of the remaining amino acids throughout the synthesis. N^(a) Boc-β-D-2NAL is prepared by a method known in the art, e.g. as described in detail in U.S. Pat. No. 4,234,571, issued Nov. 18, 1980. The side chain of D-Arg is protected with Tos. Fmoc is used as a side-chain protecting group for the amino group of Lys; whereas Z is used to protect the side-chain amino group of Dpr. The carboxyl side chain of Glu is protected with OFm, whereas the carboxyl side chain of Asp is protected with OBzl. N^(a) Boc-β-D-2NAL is introduced as the final amino acid. Boc-D-Arg(Tos), which has low solubility in CH₂ Cl₂, is coupled using a DMF:CH₂ Cl₂ mixture.

Following the assembly of a hexapeptide intermediate on a MBHA resin having the formula: Boc-Glu(OFm)-D-Arg(Tos)-Leu-Lys(Fmoc)-Pro-Dpr(Z)-MBHA resin support. The deprotection of the Glu and Lys residues and initial cyclization are carried out by treatment with 50 ml of 20 volume % piperidine in DMF for 1 hour at about 22° C., followed by washing. Then the peptidoresin, about 5 meq. (2.2 gm) of BOP [Benzotriazolyl-N-oxytris(dimethylamino)-phosphonium hexafluorophosphate] and 15 meq. of diisopropylethylamine are suspended and stirred for 2 hours at room temperature to effect cyclization between the side chains of Glu and Lys. The peptidoresin is filtered and then washed with DMF, MeOH, CH₂ Cl₂ and MeOH. Thereafter, the Boc-protecting group is removed from the cyclic hexapeptide, and the synthesis of the decapeptide is completed to produce the intermediate: ##STR10## resin support.

After deblocking the alpha-amino group at the N-terminus using trifluoroacetic acid(TFA), acetylation is achieved using a large excess of acetic anhydride in dichloromethane. Thereafter, about 4 g. of protected-peptidyl resin is suspended at room temperature in 40 ml of DMF, and 1 ml. of anhydrous hydrazine (30-40× excess) is added to it under continuous stirring. Nitrogen is bubbled through the reactive mixture, and continuous stirring is effected in a closed vial for 48 hours. The resin is filtered, washed with DMF, MeOH, CH₂ Cl₂ and MeOH, and finally dried.

About 4 g. of the protected peptide-hydrazide-resin is treated with 10-15 ml of distilled HF, in the presence of 1.5 ml of anisole as a scavenger, at 0° C. for 60 minutes to remove the remaining protecting groups and cleave the peptide from the resin. HF is removed under high vacuum, and the peptide is precipitated with anhydrous ethyl ether. The solid is collected, dissolved in 50 ml CH₃ CN:H₂ O (1:1) and lyophilized. It is then purified using RP-HPLC prior to final cyclizing.

1000 mg of the peptide-hydrazide is dissolved in 15 ml of DMF, cooled to -25° C., and N₂ gas is bubbled therethrough. 0.56 ml (about 2.25 mmol) of 4N HCl in dioxane is added, and finally 105 μl (about 0.78 mmol) of isoamylnitrite is added over ten minutes. Stirring at -25° C. is continued for 3 hours. The azide-solution is diluted with 1000 ml of precooled DMF (-25° C.); N,N diisopropylethylamine is added in suitable portions to give a final pH of 7.8. The pH is checked and readjusted several times.

The solution is stored at 4° C. for 3 days, then evaporated to dryness in high vacuum. The residue is triturated in the presence of ethyl ether. The solid is collected and dried in vacuum.

Final purification of the peptide is then effected by two RP-HPLC separations. The first preferably uses a TEAP (triethylammonium phosphate) buffer system, and the second uses a TFA buffer system, as described in detail in the J. Chromatography article.

The peptide is judged to be homogeneous using thin layer chromatography and several different solvent systems, as well as by using reversed-phase high pressure liquid chromatography and an aqueous triethylammonium phosphate solution plus acetonitrile. Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared structure, showing substantially integer-values for each amino acid in the chain. The optical rotation of Peptide No. 1 is measured on a photoelectric polarimeter as [α]_(D) ²² =-49.5°±1(c=1, 50% acetic acid).

When a peptide, such as No. 4, has a second cyclizing bond in the form of a disulfide bond between the side chains of the residues in the 4- and 10-positions, this bond may be made by air-oxidation of the cyclic decapeptide after its removal from the resin. As an example of a suitable procedure, the intermediate is prepared having the formula: ##STR11## resin support.

After alpha-amino deprotection and acetylation as described above, cleavage of the peptide from the resin and deprotection of the Cys side chains is very readily effected at 0° C. with HF, with anisole added as a scavenger prior to HF treatment. After the removal of HF under vacuum, the resin is extracted with 50% acetic acid, and the washed peptide is then air-oxidized for about 24 hours at about 22° C. to create a disulfide linkage between the Cys residues in each molecule. Finally, lyophilization is carried out to provide a crude peptide powder.

The peptides are assayed in vivo and are also tested in vitro using dissociated rat pituitary cells maintained in culture for 4 days prior to the assay. The levels of LH mediated in response to the application of peptides is assayed by specific radioimmunoassay for rat LH. Control dishes of cells only receive a measure which is 3 nanomolar in GnRH; experimental dishes receive a measure 3 nanomolar in GnRH plus a measure having either the present standard antagonist for comparison purposes i.e. [Ac-dehydroPro¹, (4F)D-Phe², D-Trp³,6 ]-GnRH or the test peptide, in concentrations ranging from 0.01 to 10 nanomolar. The amount of LH secreted in the samples treated only with GnRH is compared with that secreted by the samples treated with the peptide plus GnRH. The ability of the test peptide to reduce the amount of LH released by 3 nanomolar GnRH is compared to that of the present standard peptide.

In vivo testing determines effectiveness to prevent ovulation in female rats. In this test, a specified number of mature female Sprague-Dawley rats, e.g. five to ten, each having a body weight from 225 to 250 grams, is injected with a specified microgram dosage of peptide in either saline or bacteriostatic water at about noon on the day of proestrous. Proestrous is the afternoon of ovulation. A separate female rat group is used as a control to which the peptide is not administered. Each of the control female rats ovulates on the evening of proestrous; of the rats treated, the number of them which ovulate is recorded. Peptide No. 1 is considered to be significantly effective to prevent ovulation of female rats at a very low dosage; it is considered to be totally effective at a dose of about 500 micrograms Kg of body weight and may be effective at about 100 μg/Kg.

All peptides listed in Table I are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration, and all are considered to be effective to prevent ovulation of female mammals at reasonable dosages.

EXAMPLE II

Peptides as indicated in TABLE II having the formula: ##STR12## are prepared by the solid-phase procedure referred to above.

                  TABLE II                                                         ______________________________________                                         A              R.sub.4                                                                               R.sub.5  R.sub.8                                                                             R.sub.10                                   ______________________________________                                         17     4F          Cys    Glu    Lys  Cys                                      18     "           Dpr    "      Hly  Asp                                      19     4Cl         Orn    "      Hhl  "                                        20     "           Dbu    Hgl    Lys  Glu                                      21     "           "      "      Hly  Asp                                      22     "           Dpr    "      Hhl  "                                        23     4Br         "      Hhg    "    Glu                                      24     "           Dbu    "      Lys  "                                        25     H           "      "      Hly  Asp                                      26     4NO.sub.2   "      Glu    "    "                                        27     "           Asp    "      Hhl  Dpr                                      28     2,4Cl.sub.2 "      "      "    Dbu                                      29     "           Glu    Hgl    "    "                                        30     C.sup.a Me/4Cl                                                                             Abu    "      Lys  Cys                                      31     3,4Cl.sub.2 Cys    "      Hhl  Abu                                      ______________________________________                                    

In vitro and/or in vivo testing of the peptides specified in Table II shows that the peptides listed in Table II are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE III

Peptides as indicated in TABLE III having the formula: ##STR13## are prepared by the solid-phase procedure referred to above.

                  TABLE III                                                        ______________________________________                                         X          R.sub.4    R.sub.6     R.sub.10                                     ______________________________________                                         32      Ac     Cys        D--Arg    Abu                                        33      Acr    Abu        D--Tyr    Abu                                        34      For    Asp        "         Dpr                                        35      Bz     "          D--Arg    Dbu                                        36      Ac     Dpr        D--His    Asp                                        37      Vac    "          (Et.sub.2)D--Har                                                                         Glu                                        38      Acr    Orn        (4gua)D--Phe                                                                             "                                          39      Ac     Dbu        D--3PAL   Asp                                        40      Acr    Cys        D--His    Cys                                        41      Ac     Orn        (Et.sub.2)D--Arg                                                                         Asp                                        42      "      Dpr        D--2PAL   "                                          43      Vac    "          (4NH.sub.2)D--Phe                                                                        Glu                                        44      Bz     Glu        D--Har    Dpr                                        ______________________________________                                    

Peptides such as Nos. 32 and 33 are synthesized by employing the general teaching of U.S. Pat. No. 4,161,521.

In vitro and/or in vivo testing of the peptides specified in Table III shows that the peptides listed in Table III are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE IV

Peptides as indicated in TABLE IV having the formula: ##STR14## are prepared by the solid-phase procedure referred to above.

                  TABLE IV                                                         ______________________________________                                         R.sub.1     R.sub.4 R.sub.7    G       R.sub.10                                ______________________________________                                         45  β-D--2NAL                                                                             Asp     Nle      H       Dpr                                   46  (1Ac)D--Trp "       Met      OH      "                                     47  (6Br)D--Trp "       Tyr      H       Dbu                                   48  (5F)D--Trp  "       Nle      dehydro "                                     49  (6NO.sub.2)D--Trp                                                                          "       Met      "       Dbu                                   50  (5Cl)D--Trp "       Tyr      H       Dpr                                   51  (4Cl)D--Phe "       Phe      "       Orn                                   52  (4F)D--Phe  "       4F--D--Phe                                                                              "       "                                     53  (2,4Cl.sub.2)D--Phe                                                                        Glu     NML      OH      "                                     54  dehydroPro  "       Nle      "       Dbu                                   55  β-D--2NAL                                                                             "       Trp      H       "                                     56  (60CH.sub.3)D--Trp                                                                         "       Leu      "       Dpr                                   57  (5NH.sub.2)D--Trp                                                                          "       Nva      "       "                                     58  (4NO.sub.2)D--Phe                                                                          "       NML      "       "                                     59  dehydroPro  "       Tyr      OH      "                                     ______________________________________                                    

All peptides listed in Table IV are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration.

All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE V

Peptides as indicated in TABLE V having the formula: ##STR15## are prepared by the solid-phase procedure referred to above.

                                      TABLE V                                      __________________________________________________________________________     R.sub.1    R.sub.3 R.sub.4                                                                            R.sub.6   R.sub.10                                      __________________________________________________________________________     60 β-D--2NAL                                                                         (6NO.sub.2)D--Trp                                                                      Cys D--Arg    Cys                                           61 "       (5CH.sub.3)D--Trp                                                                      "   (DNP)D--His                                                                              Abu                                           62 "       (5OCH.sub.3)D--Trp                                                                     "   (4gua)D--Phe                                                                             Cys                                           63 dehydroPro                                                                             β-D--2NAL                                                                         "   (6NO.sub.2)D--Trp                                                                        "                                             64 "       β-D--1NAL                                                                         "   D--Val    Abu                                           65 β-D--2NAL                                                                         (1For)D--Trp                                                                           "   (Et.sub.2)D--Arg                                                                         "                                             66 "       (5F)D--Trp                                                                             Abu (5NH.sub.2)D--Trp                                                                        Cys                                           67 dehydroPro                                                                             (5Cl)D--Trp                                                                            "   D--Tyr    Abu                                           68 "       D--2PAL Glu D--Nle    Dbu                                           69 "       (1Ac)D--Trp                                                                            "   (4F)D--Phe                                                                               Orn                                           70 Pro     D--3PAL Asp β-D--1NAL                                                                           Dbu                                           71 (1For)D--Trp                                                                           "       "   (4NHCH.sub.3)D--Phe                                                                      Dpr                                           72 β-D--2NAL                                                                         "       Cys (Ipr.sub.2)D--Arg                                                                        Cys                                           73 "       (5NH.sub.2)D--Trp                                                                      "   (4NH.sub.2)D--Phe                                                                        Abu                                           74 β-D--1NAL                                                                         (6Br)D--Trp                                                                            "   (1For)D--Trp                                                                             Cys                                           75 (6CH.sub.3)D--Trp                                                                      D-- 4PAL                                                                               Asp D--4PAL   Dbu                                           __________________________________________________________________________

In vitro and/or in vivo testing of the peptides specified in Table V shows that the peptides listed in Table V are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE VI

Peptides as indicated in TABLE VI having the formula: ##STR16## are prepared by the solid-phase procedure referred to above.

                  TABLE VI                                                         ______________________________________                                         X       R.sub.1     R.sub.4 R.sub.6    R.sub.10                                ______________________________________                                         76  Acr     dehydroPro  Asp   D--Val     Dpr                                   77  Ac      "           Dpr   β-D--2NAL                                                                            Asp                                   78  Ac      β-D--2NAL                                                                             Cys   (MeEt)D--Arg                                                                              Cys                                   79  Acr     Pro         Glu   D--Ser(OtBu)                                                                              Orn                                   80  H       dehydroPro  Dpr   (imBzl)D--His                                                                             Asp                                   81  Bz      (4Br)D--Pre "     (5Cl)D--Trp                                                                               Glu                                   82  "       D--pGlu     "     (6Br)D--Trp                                                                               "                                     83  For     β-D--1NAL                                                                             "     D--Nva     Asp                                   84  "       dehydroPro  Cys   (Ipr.sub.2)D--Har                                                                         Abu                                   85  Vac     β-D--2NAL                                                                             "     D--Pro     Cys                                   86  "       D--Phe      Dbu   D--Ile     Asp                                   87  H       dehydroPro  "     D--Ala     Glu                                   ______________________________________                                    

In vitro and/or in vivo testing of the peptides specified in Table VI shows that the peptides listed in Table VI are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE VII

Peptides as indicated in TABLE VII having the formula: ##STR17## are prepared by the solid-phase procedure referred to above.

                                      TABLE VII                                    __________________________________________________________________________     R.sub.1     R.sub.3  R.sub.4                                                                            R.sub.6 R.sub.10                                      __________________________________________________________________________     88  dehydroPro                                                                             (6NO.sub.2)D--Trp                                                                       Asp β-D--2NAL                                                                         Dpr                                           89  "       "        "   D--Val  "                                             90  "       (6F)D--Trp                                                                              "   (4gua)D--Phe                                                                           "                                             91  "       "        "   D--Nva  "                                             92  "       (5OCH.sub.3)D--Trp                                                                      "   D--Pro  "                                             93  "       "        "   D--2PAL "                                             94  β-D--2NAL                                                                         (1Ac)D--Trp                                                                             Glu (Me.sub.2)D--Har                                                                       "                                             95  "       (1For)D--Trp                                                                            "   (5CH.sub.3)D--Trp                                                                      Dbu                                           96  dehydroPro                                                                             (6Br)D--Trp                                                                             Abu D--Nle  Cys                                           97  "       "        "   D--Leu  Abu                                           98  "       (6CH.sub.3)D--Trp                                                                       "   β-D--2NAL                                                                         Cys                                           99  "       (6NH.sub.2)D--Trp                                                                       Asp (4NH.sub.2)D--Phe                                                                      Dpr                                           100 "       (5NH.sub.2)D--Trp                                                                       "   D--Ala  "                                             __________________________________________________________________________

In vitro and/or in vivo testing of the peptides specified in Table VII shows that the peptides listed in Table VII are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE VII

Peptides as indicated in TABLE VIII having the formula: ##STR18## are prepared by the solid phase procedure referred to above.

                  TABLE VIII                                                       ______________________________________                                         R.sub.1     R.sub.4                                                                               R.sub.6      R.sub.7                                                                               R.sub.10                                ______________________________________                                         101  β-D--2NAL                                                                            Cys    (5NO.sub.2)D--Trp                                                                         Leu    Cys                                   102  "          "      D--3PAL    "      Abu                                   103  "          Abu    β-D--2NAL                                                                            "      Cys                                   104  "          "      (EtBu)D--Arg                                                                              "      Abu                                   105  dehydroPro "      β-D--2NAL                                                                            3PAL   Cys                                   106  "          Dpr    (4NO.sub.2)D--Phe                                                                         Tyr    Glu                                   107  β-D--2NAL                                                                            Dbu    D--3PAL    NML    "                                     108  "          Dpr    β-D--1NAL                                                                            4PAL   Glu                                   109  "          Orn    (imBzl)D--His                                                                             Leu    "                                     110  "          Asp    (6NO.sub.2)D--Trp                                                                         "      Dpr                                   111  "          Glu    D--Tyr     "      "                                     112  "          "      (1For)D--Trp                                                                              Phe    Dbu                                   113  "          "      (6F)D--Trp NML    "                                     114  (C.sup.a Me/                                                                              "      (4Cl)D--Phe                                                                               Nle    Orn                                        4Cl)D--Phe                                                                115  Pro        Asp    (imBzl)D--His                                                                             Met    "                                     116  dehydroPro "      (6OCH.sub.3)D--Trp                                                                        Nva    Dbu                                   117  "          "      (5CH.sub.3)D--Trp                                                                         "      Dpr                                   118  "          "      (1Ac)D--Trp                                                                               (4F)Phe                                                                               "                                     119  "          "      D--Nle     NML    Dbu                                   120  "          Cys    D--Arg     Nle    Abu                                   121  "          "      β-D--2NAL                                                                            Trp    Cys                                   122  Pro        Dpr    (2,4Cl.sub.2)D--Phe                                                                       Nva    Asp                                   123  β-D--2NAL                                                                            "      β-D--1NAL                                                                            Tyr    "                                     124  "          Dbu    (5Cl)D--Trp                                                                               Met    "                                     125  (4Cl)D--Phe                                                                               "      (4Br)D--Phe                                                                               3PAL   "(ace-                                                                         tate                                                                           salt)                                 ______________________________________                                    

The peptides described in TABLE VIII are tested in vivo to determine their effectiveness to prevent ovulation in female rats. All of them are considered to prevent ovulation of female rats at a low dosage, and to be totally effective at a dose of about 500 micrograms.

EXAMPLE IX

Peptides as indicated in TABLE IX having the formula: ##STR19## are prepared by the solid-phase procedure referred to above.

                  TABLE IX                                                         ______________________________________                                                R.sub.1                                                                               R.sub.3      R.sub.4                                                                               R.sub.10                                     ______________________________________                                         126      D--Glu   D--Lys       Glu  Dbu                                        127      D--Dbu   D--Hgl       "    Orn                                        128      D--Cys   D--Abu       Asp  Dbu                                        129      D--Asp   D--Orn       "    Dpr                                        130      "        D--Hly       Cys  Cys                                        131      D--Glu   D--Lys       "    Abu                                        132      D--Hhg   D--Hhl       Orn  Asp                                        133      D--Cys   D--Cys       Asp  Dbu                                        134      D--Dpr   D--Asp       Cys  Cys                                        135      D--Hly   D--Glu       "    Abu                                        136      D--Orn   D--Asp       Orn  Asp                                        137      D--Hhl   D--Hhg       "    "                                          138      D--Abu   D--Abu       Asp  Dpr                                        139      D--Abu   D--Cys       "    "                                          140      D--Hgl   D--Orn       Abu  Cys                                        ______________________________________                                    

The tricyclic peptides are made as generally explained hereinbefore through the steps of synthesizing the peptidoresin containing the cyclic hexapeptide, as set forth in Example I. Then the appropriate last 4 residues are added. For example, with respect to Peptide No. 126, after the Boc-protecting group is removed from the cyclic hexapeptide, the synthesis of the decapeptide is completed to produce the intermediate: Boc-D-Glu(OFm)-(4Cl)D-Phe-D-Lys(Fmoc)-Glu(OBzl)-Glu-β-D-2NAL-Leu-Lys-Pro-Dbu(Z)-MBHA resin support.

Following the assembly of the complete decapeptide intermediate on the MBHA resin, deprotection and cyclization of the D-Glue and D-Lys residues is carried out by treatment in the same manner as the deprotection and cyclization of Glu and Lys residues, i.e., treatment with piperidine, followed by washing, followed by treatment with BOP and diisopropylethylamine to effect cyclization between the side chains of D-Glu and D-Lys. The peptidoresin is filtered, washed with DMF, MeOH, CH₂ Cl₂ and MeOH.

After deblocking the alpha-amino group at the N-terminus using trifluoroacetic acid(TFA), acetylation is achieved using a large excess of acetic anhydride in dichloromethane. Thereafter, the protected-peptidyl resin is suspended at room temperature in DMF, and anhydrous hydrazine is added to it under continuous stirring while nitrogen is bubbled therethrough. After 48 hours, the resin is filtered, washed with DMF, MeOH, CH₂ Cl₂ and MeOH, dried and treated with distilled HF, in the presence of anisole as a scavenger, to remove the remaining protecting groups and cleave the peptide from the resin. After removal of HF, the peptide is precipitated with anhydrous ethyl ether, collected, and lyophilized. It is then purified using RP-HPLC, as in Example I, prior to final cyclizing, which is effected as set forth in Example I to achieve the third amide bond. Final purification of the tricyclic peptide is then effected as set forth in Example I.

In making a peptide, such as No. 133, the disulfide bond between the D-Cys residues in the 1- and 3-positions is preferably effected by oxidation prior to conversion of the linear peptide to the azide.

In vitro and/or in vivo testing of the peptides specified in Table IX shows that the peptides listed in Table IX are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE X

Bicyclic peptides as indicated in TABLE X having the formula: ##STR20## with a second cyclizing bond between either R₅ and R₈ or R₄ and R₁₀ are prepared by the solid-phase procedure referred to above.

                  TABLE X                                                          ______________________________________                                                R.sub.5                                                                             R.sub.8       R.sub.4                                                                               R.sub.10                                      ______________________________________                                         141      Glu    Lys           Ser  D--Ala                                      142      "      Hly           "    Gly                                         143      Hgl    "             Orn  "                                           144      "      Hhl           Dbu  D--Ala                                      145      Hhg    "             Dpr  "                                           146      Tyr    Har           "    Dbu                                         147      "      (Et.sub.2)Har Abu  Cys                                         148      "      Arg           Asp  Dbu                                         149      "      (Ipr.sub.2)Har                                                                               Glu  Dpr                                         150      "      (Et.sub.2)Arg Cys  Cys                                         151      "      (Ipr.sub.2)Arg                                                                               Abu  Abu                                         152      "      Arg           Orn  Asp                                         ______________________________________                                    

In vitro and/or in vivo testing of the peptides specified in Table X shows that the peptides listed in Table X are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XI

Monocyclic peptides as indicated in TABLE XI having the formula: ##STR21## are prepared by the solid-phase procedure referred to above.

                  TABLE XI                                                         ______________________________________                                         R.sub.1     R.sub.3   R.sub.6     R.sub.10                                     ______________________________________                                         153   D--Asp    D--Lys    β-D--2NAL                                                                           D--Ala                                     154   D--Glu    "         "         "                                          155   D--Orn    D--Glu    "         Gly                                        156   D--Glu    D--Orn    "         D--Ala                                     157   Orn       Asp       D--3PAL   "                                          158   Asp       Dpr       D--Arg    "                                          159   "         Lys       β-D--2NAL                                                                           "                                          160   Glu       D--Lys    "         "                                          161   D--Abu    D--Abu    D--3PAL   Gly                                        162   D--Lys    Glu       "         "                                          163   D--Asp    Dbu       D--Leu    D--Ala                                     164   D--Glu    D--Hly    D--3PAL   D--Ala                                     165   D--Lys    D--Hgl    "         Gly                                        166   D--Glu    D--Hhl    D--Arg    D--Ala                                     167   Lys       Hgl       D--3PAL   "                                          168   Hly       "         D--Arg    "                                          169   Hhg       Hly       β -D--2NAL                                                                          "                                          170   "         Hhl       "         "                                          ______________________________________                                    

In vitro and/or in vivo testing of the peptides specified in Table XI shows that the peptides listed in Table X are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XII

Monocyclic, bicyclic and tricyclic peptides as indicated in TABLE XII having the formula: ##STR22## with the 1- and 3-position residues being in the D-isomer form, with a second cyclizing bond between either R₅ and R₈ or R₄ and R₁₀ being present in Peptides 175-179 and with second and third cyclizing bonds being present in Peptides 180-183 prepared by the solid-phase procedure referred to above.

                  TABLE XII                                                        ______________________________________                                         n           R.sub.4                                                                               R.sub.5  R.sub.8                                                                               R.sub.10                                    ______________________________________                                         171    7        Ser    Tyr    Arg    D--Ala                                    172    5        "      "      (Ipr.sub.2)Har                                                                        Gly                                       173    8        Orn    "      (Et.sub.2)Arg                                                                         "                                         174    6        Dbu    "      Arg    D--Ala                                    175    10       Ser    Glu    Lys    "                                         176    9        "      Hgl    Hly    Gly                                       177    7        Abu    Tyr    (Et.sub.2)Har                                                                         Cys                                       178    11       Asp    "      Arg    Dbu                                       179    9        Glu    "      Har    Dpr                                       180    8        Cys    Glu    Lys    Cys                                       181    7        Abu    "      Hly    Abu                                       182    11       Orn    Hgl    "      Asp                                       183    10       Glu    Hhg    Hhl    Dbu                                       ______________________________________                                    

In vitro and/or in vivo testing of the peptides specified in Table XII shows that the peptides listed in Table XII are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

The peptides of the invention are often administered in the form of pharmaceutically acceptable, nontoxic salts, such as acid addition salts, or of metal complexes, e.g., with zinc, barium, calcium, magnesium, aluminum or the like (which are considered as addition salts for purposes of this application), or of combinations of the two. Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, nitrate, oxalate, fumarate, gluconate, tannate, maleate, acetate, citrate, benzoate, succinate, alginate, malate, ascorbate, tartrate and the like. For example, an aqueous solution of the peptide can be repeatedly treated with IN acetic acid and then lyophilized to yield the acetic acid salt thereof. If the active ingredient is to be administered in tablet form, the tablet may contain a pharmaceutically-acceptable diluent which includes a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used as part of the pharmaceutically-acceptable diluent, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

The pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. Usually, the dosage will be from about 10 micrograms to about 2.5 milligrams of the peptide per kilogram of the body weight of the host when given intravenously; although oral dosages will be higher, it is anticipated that the cyclic nature of these compounds will permit more effective oral administration. Overall, treatment of subjects with these peptides is generally carried out in the same manner as the clinical treatment using other antagonists of GnRH using a suitable carrier in which the peptide is soluble.

It may also be desirable to deliver the GnRH analog over prolonged periods of time, for example, for periods of one week to one year from a single administration, and slow release, depot or implant dosage forms may be utilized. For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has a low degree of solubility in body fluids, for example, an acid addition salt with a polybasic acid; a salt with a polyvalent metal cation; or combination of the two salts. A relatively insoluble salt may also be formulated in a gel, for example, an aluminum stearate gel. A suitable, slow-release depot formulation for injection may also contain the GnRH analog or a salt thereof dispersed or encapsulated in a slow degrading, non-toxic or non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer, for example, as described in U.S. Pat. No. 3,773,919. These compounds may also be formulated into silastic implants.

These peptides can be administered to mammals intravenously, subcutaneously, intramuscularly, orally, percutaneously, e.g. intranasally or intravaginally to achieve fertility inhibition and/or control and also in applications calling for reversible suppression of gonadal activity, such as for the management of precocious puberty or during radiation- or chemo-therapy. They are also useful for treatment of steroid-dependent tumors. Effective dosages will vary with the form of administration and the particular species of mammal being treated. An example of one typical dosage form is a bacteriostatic water solution containing the peptide which solution is administered to provide a dose in the range of about 0.1 to 2.5 mg/kg of body weight. Oral administration of the peptide may be given in either solid form or liquid form.

Results of in vivo testing of selected of the foregoing antagonists are shown in the following Table B:

                  TABLE B                                                          ______________________________________                                         Peptide  Dosage  Rats        Dosage                                                                               Rats                                        No.      μg   Ovulating   μg Ovulating                                   ______________________________________                                          1.      50      0/7          25   1/7                                                  10      0/10         5     2/10                                                2.5     9/10                                                           3.      100     1/8          50   2/8                                         153.     500     0/7         100   0/5                                                  25      1/11         10   2/8                                         154.     500     0/7         100   0/7                                         156.     100     0/10         50   0/8                                                  25      5/10                                                          158.     500     3/5         100   4/5                                         ______________________________________                                    

Although the invention has been described with regard to its preferred embodiments, it should be understood that changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims which are appended hereto. For example, the use of cyclizing bond between the residues in positions 1 and 3 may produce biologically potent peptides without the presence of an accompanying second bond. Other substitutions known in the art which do not significantly detract from the effectiveness of the peptides may be employed in the peptides of the invention. D-2PAL and D-4PAL are considered to be equivalents of D-3PAL. Substituted Phe, such as (4F)Phe, can be used instead of Phe in the 7-position; substituted Phe, such as (2Cl)Phe, can be used instead of Tyr in the 5-position. Other hydrophobic amino acid residues can also be employed in the 1-position, preferably in D-isomer form, and are considered equivalents of those specified.

Particular features of the invention are emphasized in the claims which follow. 

What is claimed is:
 1. A peptide or a nontoxic salt thereof, said peptide having the formula: ##STR23## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁ is dehydroPro, D-pGlu, (A)D-Phe, (B)D-Trp, Pro, or β-D-NAL; A is H,Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; R₃ is D-PAL β-D-NAL or (B)D-Trp; R₄ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₅ is Glu, Hgl or Hhg; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; C is H, imBzl or dinitrophenyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; D is H or di-lower alkyl; R₈ is Lys, Hly or Hhl; E is H, OH or dehydro and R₁₀ is cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R.sub. 4 is Cys or Abu, R₁₀ is Cys or Abu; when R₄ is Asp or Glu, R₁₀ is Orn, Dbu or Dpr; and when R₄ is Orn, Dbu or Dpr, Re is Asp or Glu.
 2. A peptide in accordance with claim 1 wherein A is 4Cl or 4F, R₅ is Glu and R₈ is Lys.
 3. A peptide in accordance with claim 2 wherein R₁ is β-D-2NAL, R₃ is D-PAL and R₇ is Leu.
 4. A peptide in accordance with claim 3 wherein R₆ is D-Trp, D-PAL, β-D-2NAL, (imBzl)D-His or (6NO₂)D-Trp.
 5. A peptide in accordance with claim 1 wherein R₄ is Asp or Glu and R₁₀ is Orn, Dbu or Dpr.
 6. A peptide in accordance with claim 1 wherein R₄ is Orn, Dbu or Dpr and R₁₀ is Asp or Glu.
 7. A peptide in accordance with claim 1 wherein R₄ is Cys and R₁₀ is Cys or Abu.
 8. A peptide in accordance with claim 1 wherein R₃ is (B)D-Trp and B is 6NO₂ or N^(in) For.
 9. A peptide in accordance with claim 8 wherein R₄ is Cys or Abu and R₁₀ is Cys.
 10. A peptide in accordance with claim 1 wherein R₄ is Abu and R₁₀ is Abu.
 11. A peptide in accordance with claim 1 wherein R₄ is Asp and R₁₀ is Dpr.
 12. A peptide in accordance with claim 11 wherein X is Ac, A is 4Cl or 4F and E is H.
 13. A peptide in accordance with claim 12 wherein R₁ is B-D-2NAL and R₆ is D-Arg.
 14. A peptide in accordance with claim 13 wherein R₃ is D-Trp and R₇ is Leu.
 15. A peptide in accordance with claim 1 wherein R₁ is dehydroPro, R₃ is D-PAL, R₄ is Glu and R₁₀ is Dpr.
 16. A peptide in accordance with claim 1 having the formula ##STR24##
 17. A peptide in accordance with claim 1 having the formula ##STR25##
 18. A peptide in accordance with claim 1 having the formula ##STR26##
 19. A method for regulating the secretion of gonadotropins comprising administering an effective amount of a peptide or a nontoxic salt thereof as defined in claim
 1. 20. A peptide or a nontoxic salt thereof, said peptide having the formula: ##STR27## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R is D-Cys, D-Abu, D-Asp, D-Glu,D-Hgl, D-Hhg, D-Lys, D-Hly, D-Hhl, D-Orn, D-Dbu or D-Dpr; A is H,Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; R₃ is D-Cys, D-Abu, D-Asp, D-Glu, D-Hgl, D-Hhg, D-Lys, D-Hly, D-Hhl, , D-Orn, D-Dbu or D-Dpr; R₄ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₅ is Glu, Hgl or Hhg; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; C is H, imBzl or dinitrophenyl; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; R₈ is Lys, Hly or Hhl; E is H, OH or dehydro and R₁₀ is Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R₄ is Cys or Abu, R₁₀ is Cys or Abu; when R₄ is Asp or Glu, R₁₀ is Orn, Dbu or Dpr; and when R₄ is Orn, Dbu or Dpr, R₁₀ is Asp or Glu and provided further that when R₁ is D-Cys or D-Abu, R₃ is D-Cys or D-Abu; when R₁ is D-Asp, D-Glu, D-Hgl or D-Hhg, R₃ is D-Lys, D-Hly, D-Hhl, D-Orn, D-Dbu or D-Dpr; and when R₁ is D-Lys, D-Hly, D-Hhl, D-Orn, D-Dbu or D-Dpr, R₃ is D-Asp, D-Glu, D-Hgl or D-Hhg.
 21. A peptide in accordance with claim 20 wherein A is 4Cl or 4F, R₅ is Glu, R₇ is Leu and R₈ is Lys.
 22. A peptide in accordance with claim 21 wherein R₆ is D-Trp, D-PAL, β-D-2NAL, (imBzl)D-His or (6NO₂)D-Trp.
 23. A peptide in accordance with claim 20 wherein R₄ is Asp or Glu and R₁₀ is Orn, Dbu or Dpr.
 24. A peptide in accordance with claim 20 wherein R₄ is Orn, Dbu or Dpr and R₁₀ is Asp or Glu.
 25. A peptide in accordance with claim 20 wherein R₄ is Cys and R₁₀ is Cys or Abu.
 26. A peptide in accordance with claim 20 wherein R₄ is Asp and R₁₀ is Dpr.
 27. A peptide in accordance with claim 26 wherein R₁ is D-Asp and R₃ is D-Lys or D-Orn.
 28. A peptide in accordance with claim 26 wherein R₁ is D-Cys and R₃ is D-Cys.
 29. A peptide in accordance with claim 20 having the formula ##STR28##
 30. A peptide or a nontoxic salt thereof which contains two cyclizing bonds between two of the groups R₁ and R₃, R₄ and R₁₀, and R₅ and R₈, respectively, said peptide having the formula: ##STR29## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁ is β-D-2NAL, dehydroPro, D-Cys, D-Abu, D-Asp, D-Glu, D-Lys, D-Orn, D-Dbu or D-Dpr; A is H,Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; R₃ is β-D-2NAL, D-3PAL, D-Trp, D-Cys, D-Abu, D-Asp, D-Glu, D-Lys, D-Orn, D-Dbu or D-Dpr; R₄ is Ser, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₅ is Tyr, Glu, Hgl or Hhg; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; C is H imBzl or dinitrophenyl; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; R₈ is (D)Arg, (D)Har, Hly, Hhl or Lys; E is H, OH or dehydro; and R₁₀ is Gly, D-Ala, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R₄ is Cys or Abu, R₁₀ is Cys or Abu; when R₄ is Asp or Glu, R₁₀ is Orn, Dbu or Dpr; when R₄ is Orn, Dbu or Dpr, R₁₀ is Asp or Glu; when R₅ is Glu, R₈ is Lys; when R₁ is D-Cys or D-Abu, R₃ is D-Cys or D-Abu; when R₁ is D-Asp or D-Glu, R₃ is D-Lys, D-Orn, D-Dbu or D-Dpr; and when R₁ is D-Lys, D-Orn, D-Dbu or D-Dpr, R₃ is D-Asp or D-Glu.
 31. A peptide or a nontoxic salt thereof which contains a cyclizing bond between R₁ and R₃ and may contain one or two additional cyclizing bonds between the groups R₄ and R₁₀, and R₅ and R₈, respectively, said peptide having the formula ##STR30## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R₁ is D-Cys, D-Abu, asp, glu, hgl, hhg, lys, hly, hhl, orn, dbu or dpr; A is H,Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; R₃ is D-Cys, abu, asp, glu, hgl, hhg, lys, hly, hhl, orn, dbu or dpr; R₄ is Ser, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₅ is Tyr, Glu, Hgl or Hhg; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; C is H, imBzl or dinitrophenyl; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; R₈ is (D)Arg, (D)Har, Lys, Hly or Hhl; E is H, OH or dehydro; and R₁₀ is Gly, D-Ala, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; provided however that when R₄ is Cys or Abu, it is bonded to R₁₀ which is Cys or Abu; when R₄ is Asp or Glu, it is bonded to R₁₀ which is Orn, Dbu or Dpr; when R₄ is Orn, Dbu or Dpr, it is bonded to R₁₀ which is Asp or Glu; when R₅ is Glu, Hgl or Hhg, it is bonded to R₈ which is Lys, Hly or Hhl; when R₁ is D-Cys or D-Abu, it is bonded to R₃ which is D-Cys or D-Abu; when R₁ is asp, glu, hgl or hhg, it is bonded to R₃ which is lys, hly, hhl, orn, dbu or dpr; and when R₁ is lys, hly, hhl, orn, dbu or dpr, it is bonded to R₃ which is asp, glu, hgl or hhg.
 32. A peptide in accordance with claim 31 wherein R₁ is D-Glu or D-Hgl and R₃ is D-Lys or D-Hly.
 33. A peptide in accordance with claim 31 wherein R₁ is D-Cys and R₃ is D-Cys.
 34. A peptide in accordance with claim 31 wherein R₁ is D-Abu and R₃ is D-Abu.
 35. A cyclic peptide or a nontoxic salt thereof which may contain one or two additional cyclizing bonds between two of the groups R₄ and R₁₀, and R₅ and R₈, respectively, said peptide having the formula: ##STR31## wherein X is hydrogen or an acyl group having 7 or less carbon atoms; A is H,Cl, F, NO₂, CH₃, OCH₃, C^(a) Me/4Cl, Cl₂ or Br; n=4 to 11; R₄ is Ser, Cys, Asp, Glu, Orn, Dbu, Dpr or Abu; R₅ is Tyr, Glu, Hgl or Hhg; R₆ is β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Har, D-Tyr, (C)D-His, D-PAL, (D)D-Arg, D-Leu, D-Ile, D-Val, D-Nle, D-Ala, D-Pro, D-Ser(OtBu), or D-Nva; A' is A, NH₂, NHCH₃ or gua; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; C is H, imBzl or dinitrophenyl; D is H or di-lower alkyl; R₇ is Nle, Leu, NML, Phe, Met, Nva, Tyr, Trp or PAL; E is H, OH or dehydro; R₈ is (D)Arg, (D)Har, Lys, Hly or Hhl and R₁₀ is Gly D-Ala Cys Asp Glu Orn Dbu Dpr or Abu; provided however that when R₄ is Cys or Abu, it is bonded to R₁₀ which is Cys or Abu; when R₄ is Asp or Glu, it is bonded to R₁₀ which is Orn, Dbu or Dpr; when R₄ is Orn, Dbu or Dpr, it is bonded to R₁₀ which is Asp or Glu; and when R₅ is Glu, Hgl or Hhg, it is bonded to R₈ which is Lys, Hly or Hhl. 